A. Field of the Invention
The present invention relates to the field of gene expression, generally, and more specifically it adduces the expression recombinant transgenes in host cells. The invention may be exploited in the production of recombinant proteins in vitro or in gene therapy in vivo.
B. Related Art
The ability to express foreign genes in host cells has become a pivotal tool in molecular biology. For example, expressing proteins in host cells in vitro can lead to the large scale production of the protein for use in research or therapy. Examples of proteins that could be used in this manner are hormones, such as insulin, or cytokines, such as the interleukins. Scientists are constantly seeldng ways to maximize expression of tansgenes when they are imported into host cells.
Another important technology affected by foreign gene expression is gene therapy. Attaining high level expression in specific target cells is a key aspect of gene therapy, too. Numerous parameters have been varied in an effort to achieve higher levels of expression including the mode of gene trasduction, the vector, the promoter, as well as the dose and routes of administration.
Recently Son & Huang (1994) reported that exposure of CDDP-resistant ovarian carcinoma cells to CDDP prior to liposome-mediated gene transfer resulted in enhanced gene transduction. This study utilized an ovarian cancer cell line, 2008, that rapidly acquire CDDP-resistance following exposure. The cells in this study were exposed to CDDP for four to six weeks prior to gene transfer (in vitro) or exposed once, one week prior to gene transfer (in vivo). According to the authors, their data only indicate that CCDP-resistant cells show improved gene transduction. Thus, from this study, it is unclear whether CDDP-sensitive cells would provide the same results. It also is unclear whether the effect was tied to liposomal transfection methods, or could be more broadly applied.
A similar phenomenon has been observed in primary human foreskin fibroblasts infected with adeno-associated virus following brief exposure to high concentrations of CDDP or other DNA-damaging agents (Alexander et al., 1994; Russell et al., 1995). In this system, these non-malignant cells were rendered more susceptible to transduction by sublethal, but relatively high levels of DNA damaging agents. The treatments were conducted for 16 to 20 hours, followed immediately by transduction. The cause of the increased expression was not correlated with other transduction methodologies. Moreover, it was unclear from the data whether the treatment rendered the cells more competent for AAV transduction, whether increased uptake of AAV was induced or whether increased expression of AAV occurred following internalization.
Thus, it is clear that the prior art does not provide a clear picture, with respect to the effect of DNA-damaging agents, of their effects on the transduction and expression of transgenes in various host cells. There remains a need for a better understanding of these phenomena and for increasing the expression of transgenes in transduced cells.